Fluidic structures including meandering and wide channels

ABSTRACT

The present invention relates generally to microfluidic structures, and more specifically, to microfluidic structures and methods including meandering and wide channels. Microfluidic systems can provide an advantageous environment for performing various reactions and analyses due to a reduction in sample and reagent quantities that are required, a reduction in the size of the operating system, and a decrease in reaction time compared to conventional systems. Unfortunately, the small size of microfluidic channels can sometimes result in difficulty in detecting a species without magnifying optics (such as a microscope or a photomultiplier). A series of tightly packed microchannels, i.e., a meandering region, or a wide channel having a dimension on the order of millimeters, can serve as a solution to this problem by creating a wide measurement area. Although this invention mainly describes the use of meandering and wide channels in heterogeneous immunoassays on a microfluidic chip, this invention could be used for amplifying optical signals for other types of reactions and/or assays.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/887,487, entitled “Fluidic Structures Including Meandering and WideChannels” filed Feb. 24, 2010, which is a U.S. National Application ofInternational Application No. PCT/US06/014583, entitled “FluidicStructures Including Meandering and Wide Channels” filed Apr. 19, 2006,which claims benefit under 35 U.S.C. §119(e) of U.S. ProvisionalApplication Ser. No. 60/672,921, entitled “Fluidic Structures IncludingMeandering and Wide Channels” filed Apr. 19, 2005, each of which ishereby incorporated by reference in its entirety.

This invention was made with Government support under GM065364 awardedby National Institutes of Health and under ESC-0004030 awarded byNational Science Foundation. The Government has certain rights in theinvention.

FIELD OF INVENTION

The present invention relates generally to microfluidic structures, andmore specifically, to microfluidic structures and methods includingmeandering and wide channels.

BACKGROUND

Assays such as heterogeneous immunoassays (i.e., an assay where onecomponent in the liquid phase binds with another component in the solidphase) are widely used for applications in life sciences anddiagnostics, and are usually carried out in microwells. In the microwellformat, however, long incubation times are typically required. As theaffinity reaction proceeds, the concentration of the molecules in thelayer of fluid located close to the surface decreases. Diffusion ofmolecules from the bulk of the solution is then needed to replenish thatlayer of fluid to allow more binding events to take place. Longincubation times are needed to allow the diffusion of the molecules fromthe bulk of the solution towards the surface. Recent developments showedthat these diffusion-limited reactions take place faster in channels ofmicrometer dimensions, i.e. in microfluidic devices. One reason for thefast reaction times is attributed to the presence of a flow of freshsolution next to the solid phase. Incubation under flow-conditions inmicrochips (i.e., microfluidic chips or devices) achieves fast transportof molecules to the surface, and replenishes the layer of fluid close tothe surface faster than by the diffusion mechanism.

In microfluidic channels, a small volume of solution (i.e., microlitersor less) can sustain a flow sufficient for a fast replenishment of thesolution close to the surface for several minutes. These features arequite attractive for the applications of immunoassays, because theyresult in the consumption of less solution and in faster assays comparedto the microwell format. As a result, heterogeneous immunoassays inmicrofluidic devices have been reported frequently in the scientificliterature. In these reports, the lateral dimensions of the channelswere typically around 10-200 μm. These dimensions are well suited tobenefit from the advantage of microfluidics for immunoassays, but theyrequire the use of magnifying optics and the precise positioning ofoptics to allow detection of a signal within the channel. Thesetechniques typically require substantial capital equipment that can beboth expensive and bulky, thus limiting where and when the detection cantake place. Advances in the field that could, for example, reduce costsand/or increase portability would find application in a number ofdifferent fields.

SUMMARY OF THE INVENTION

Fluidic (e.g., microfluidic) structures and methods associated therewithare provided.

In one aspect of the invention, a device is provided. The devicecomprises a microfluidic channel comprising a meandering region definedby an area of at least 0.5 mm², wherein at least 50% of the area of themeandering region comprises an optical detection pathway.

In another aspect of the invention, a method is provided. The methodcomprises exposing a meandering microfluidic channel defined by an areaof at least 0.5 mm² to light, and measuring a signal over a measurementarea comprising more than one adjacent segments of the meanderingchannel.

In another aspect of the invention, a method is provided. The devicecomprises a microfluidic channel comprising a meandering region, whereinat least 50% of the area of the meandering region comprises an opticaldetection pathway, and a detector aligned with the optical detectionpathway, the detector able to detect a signal within at least 50% of thearea of the meandering region.

In another aspect of the invention, a method is provided. The methodcomprises accumulating an opaque material on a meandering region of amicrofluidic channel, exposing at least 50% of the area of themeandering region to light, and determining the transmission of lightthrough the opaque material and through at least 50% of the area of themeandering region.

In another aspect of the invention, a method is provided. The methodcomprises passing a fluid over a surface of a meandering region of amicrofluidic channel, allowing a sample component to bind with a bindingpartner disposed on the surface, allowing a metal colloid to associatewith a sample component, and flowing a metal solution over the surfaceto form a metallic layer.

In another aspect of the invention, a method is provided. The methodcomprises passing a fluid over a surface of a microfluidic channelhaving a width of at least 1 mm, wherein the channel comprises an uppersurface and a lower surface, allowing a sample component to bind with abinding partner disposed on at least one of the upper and lowersurfaces, allowing a metal colloid to associate with a sample component,and flowing a metal solution over the surface to form a metallic layer.

In another aspect of the invention, a device is provided. The devicecomprises accumulating an opaque material on a region of a microfluidicpathway for fluid flow, wherein the pathway is defined in part by anupper surface and a lower surface, the pathway having a width, a length,and a height, at least a portion of the width being at least about 1 mm,at least a portion of the length being at least about 1 mm, and at leasta portion of the height being less than or equal to about 500 μm,exposing the region to light, and determining the transmission of lightthrough the opaque material.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control. If twoor more documents incorporated by reference include conflicting and/orinconsistent disclosure with respect to each other, then the documenthaving the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying figures, which areschematic and are not intended to be drawn to scale. In the figures,each identical or nearly identical component illustrated is typicallyrepresented by a single numeral. For purposes of clarity, not everycomponent is labeled in every figure, nor is every component of eachembodiment of the invention shown where illustration is not necessary toallow those of ordinary skill in the art to understand the invention. Inthe figures:

FIGS. 1A-B show schematically a meandering channel configuration,according to one embodiment of the invention.

FIGS. 2A-B show schematically another meandering channel configuration,according to another embodiment of the invention.

FIGS. 3A-C show photographs of the results of immunoassays performed ina meandering channel configuration, according to another embodiment ofthe invention.

FIG. 4 shows graphically the results of an immunoassay based onamplification by silver enhancement, according to another embodiment ofthe invention.

FIG. 5 shows graphically the results of an immunoassay based onamplification by a precipitating dye, according to another embodiment ofthe invention.

FIG. 6 shows graphically the result of an immunoassay before and afterthe substrate is rotated relative to a detector, according to anotherembodiment of the invention.

FIGS. 7A-D show a wide channel configuration, according to anotherembodiment of the invention.

FIG. 8 shows schematically an example of a detection device and method,according to another embodiment of the invention.

FIG. 9 shows a vessel used for delivering fluids into a microfluidicdevice, according to another embodiment of the invention.

FIGS. 10A-E show schematically examples of different shapes andconfigurations of meandering channels, according to another embodimentof the invention.

DETAILED DESCRIPTION

The present invention relates generally to microfluidic structures, andmore specifically, to structures and methods including meandering andwide channels. Microfluidic systems can provide an advantageousenvironment for performing various reactions and analyses due to areduction in sample and reagent quantities that are required, areduction in the size of the operating system, and a decrease inreaction time compared to conventional systems. Unfortunately, the smallsize of microfluidic channels can sometimes result in difficulty indetecting a species without magnifying optics (such as a microscope, aphotomultiplier, or an avalanche photodiode). A series of tightly packedmicrochannels, i.e., a meandering region, or a wide microchannel havinga dimension on the order of millimeters, can serve as a solution to thisproblem by creating a wide measurement area. Although this inventionrelates to the use of meandering and wide channels in heterogeneousimmunoassays on a microfluidic chip, this invention can also be used foramplifying optical signals for other types of reactions and/or assays.

FIG. 1A shows an example of a meandering channel configuration. As usedherein, a meandering channel (i.e., a channel having a meanderingregion) includes at least a first segment that has a flow path in afirst direction and a second segment that has a flow path in a seconddirection substantially opposite (e.g., greater than 135 degrees from)the first direction. Examples of meandering channel regions are shown inFIGS. 1 and 10. Oftentimes, a meandering channel will include more thantwo alternating segments that extend in opposite directions. Channel 12includes a meandering (e.g., serpentine) region 20 that includes atightly packed channel system having a series of turns 30 that span overa large area (A) relative to width 40 of the channel. The area spannedby the meandering channel (i.e., the area of the meandering region) isthe rectangular area bound by outermost points of the meandering channelalong each axis. In FIG. 1, the area (A) that meandering channel region20 spans is defined by the surface area given by dimension 50 times (×)dimension 60. Typically, the area spanned by the channel (i.e., asviewed from above the channel, perpendicular to the direction of fluidflow) is on the order of millimeters squared (mm²). For instance, thearea may be greater than or equal to 0.5 mm², greater than or equal to 1mm², greater than or equal to 2 mm², greater than or equal to 5 mm²,greater than or equal to 10 mm², or greater than or equal to 50 mm².However, in other embodiments, e.g., depending on the method used fordetection, the area spanned by a meandering channel may be between 0.25mm² and 0.5 mm², or between 0.1 mm² and 0.25 mm².

Typically, the area spanned by the meandering channel is designed to berelatively large (e.g., on the order of mm²) compared to conventionalmicrofluidic systems, so that a wide area can be used for detection andso the total amount of signal that can be detected is increased. Forinstance, in one embodiment, a signal detected is produced in themeandering channel, and the signal spans over the entire area (e.g.,mm²) of the meandering region. Thus, the detection region may span overthe entire area of the meandering region. Conventional microfluidicsystems, however, have a detection region on the order of square microns(μm²), which is typically defined by the width of the microchannel.These conventional systems generally require magnifying optics such asmicroscopes, avalanche photodiodes, or photomultipliers, as well asprecise alignment of the magnifying optics to the microchannel, in orderto detect a species in the microchannel. In the meandering channelsystem, magnifying optics and precise alignment can be avoided since anarea on the order of mm² can be detected by the naked eye. This allows auser to read a signal (e.g., a change in color, turbidity, opacity,agglutination, etc.) directly from the microchip, and/or, as shown inFIG. 1B, to align detector 100 (e.g., an optical reader) over a seriesof segments of the channel, rather than directly over a singlemicrochannel. In one embodiment, detector 100 can be placed in a varietyof positions by the user, e.g., positions 110 and 120, without precisealignment over region 20, since the average signal obtained from thedetector in positions 110 and 120 is the same. The large area of themeandering region also allows it to be compatible with simplifiedoptical detectors (as discussed in more detail below), as well as withconventional spectrophotometers and optical readers (e.g., 96-well platereaders).

The positioning of the detector over the meandering region without theneed for precision is an advantage, since external (and possibly,expensive) equipment such as microscopes, lenses, and alignment stagesare not required. Instead, alignment can be performed by eye, or bylow-cost methods that may not require an alignment step by the user. Inone embodiment, a microchip comprising a meandering region can be placedin a simple holder (i.e., in a cavity having the same shape as themicrochip), and the measurement area can be automatically located in abeam of light of the detector. Possible causes of misalignment causedby, for instance, chip-to-chip variations, the exact location of thechip in the holder, and normal usage of the device, are negligiblecompared to the dimensions of the measurement area. As a result, themeandering region can stay within the beam of light and detection maynot be interrupted due to these variations.

Detector 100 may be aligned over meandering channel region 20 to varyingdegrees and may depend on, for instance, the number of detectors alignedover the region, the size of the detector, and/or the size of the areaof the meandering region. In one embodiment, the detector can alignwithin at least 25% of area of the meandering region. In anotherembodiment, the detector can align within at least 50% of area of themeandering region. In yet another embodiment, the detector can alignwithin at least 75% of area of the meandering region. A small areacovered by the detector (e.g., 25% or less of the area of the meanderingregion) may be suitable for aligning several detectors over the region(e.g., for determining different signals within different portions ofthe channel within the region). A large area covered by the detector(e.g., 75% or more of the area of the meandering region) may occur whenaligning a single detector over the region (e.g., if the detector andthe region are of similar size).

The detector may detect a signal within all, or a portion, of themeandering region. In other words, different amounts of the meanderingregion may be used as an optical detection pathway. For instance, thedetector may detect a signal within at least 25% of the meanderingregion, within at least 50% of the meandering region, or within at least75% of the meandering region. In some instances, 100% of the meanderingregion is used for detection by a detector (e.g., detection in atransparent channel by the unaided eye). The area in which themeandering region is used as an optical detection pathway may alsodepend on, for instance, the opacity of the material in which thechannel is fabricated (e.g., whether all, or, a portion, of the channelis transparent), the amount of a non-transparent material that may covera portion of the channel (e.g., via use of a protective cover), and/orthe size of the detector and the meandering region.

The spacing of adjacent channel segments 70 and 75 (FIG. 1A) of themeandering channel can vary depending on the application. In someembodiments, segments 70 and 75 are separated by a distance of less than1 mm, less than 500 μm, less than 250 μm, less than 100 μm, less than 50μm, less than 25 μm, less than 10 μm, less than 5 μm, less than 1 μm, orless than 0.1 μm. For example, segments 70 and 75 may be separated by adistance of less than 0.01 times, less than 0.1 times, less than 0.25times, less than 0.5 times, less than 1 times, less than 2 times, lessthan 5 times, or less than 10 times channel width 40. In some particularembodiments, observable gaps may not exist between segments, e.g., twoadjacent segments may appear as a single segment. In some instances, itmay be desirable to place adjacent segments close together (e.g.,separated by less than 0.25 times channel width 40) so that more of asignal can be generated over a given area (i.e., less area is covered bynon-channel regions). In other cases, adjacent segments may be placedfarther apart (e.g., separated by greater than 5 times channel width30). This can be useful, for example, if the concentration of detectablespecies in the channel is high, if the detectable signal gives a binary(e.g., yes/no) response, and/or if it is desirable to have a channelhaving a relatively short length.

As shown in FIG. 1, the length of segments 70 and 75 are the same. Inother embodiments, however, the lengths of the segments of themeandering channel vary within the channel. For instance, as shown inFIG. 2, segments 80 and 85 have different lengths, since meanderingchannel 15 has a circular shape. The meandering channel (and the area ofthe channel) can be designed to have any shape, e.g., a square,rectangular, circular, oval, triangular, spiral, or an irregular shape,since the overall shape does not affect the fluid flow conditions withinthe channel, as discussed in more detail below.

The meandering channels are not limited by the number and/or the degreeof turns that make up the channel. For instance, in FIG. 1 turn 30 is a180 degree turn, and meandering channel region 20 is made up of 25 ofthese turns. In FIG. 2, turn 35 is also a 180 degree turn, andmeandering channel 15 is made up of 34 of turns 35. Meandering channelshaving areas of different shapes, e.g., a spiral shape, and otherconfigurations are also possible (FIG.10).

The meandering channel has at least one cross-sectional dimension in themicron range so that it can retain its advantageous qualities ofmicro-scale geometry (e.g., laminar flow, fast reaction times, smallvolumes, etc.). For example, the cross-sectional dimension may be lessthan 1 mm, less than 500 μm, less than 250 μm, less than 100 μm, lessthan 50 μm, or less than 25 μm. In some instances, it is desirable tohave a channel having a relatively large cross-sectional dimension(e.g., a height of 500 μm), for example, to increase the path length ofthe channel; this configuration may be useful for detecting a weaksignal that depends on the amount of detectable species solvated orsuspended in the fluid (e.g., colorimetric detection), rather than theamount of detectable species formed on the surface of the channel. Insome cases, a channel may be designed to have a width greater than about75 μm, so that gas bubbles can pass easily through the channel, asdescribed below. In other cases, a small cross-sectional dimension(e.g., 50 μm or less) is suitable, e.g., for flowing very small amountsof fluid in the device. A cross-sectional dimension in the micron rangealso allows transport of fresh solution over the surface of the channelwithout the use of large amounts of fluid.

Different types of binding, including those that involve chemical and/orbiological reactions, may take place in a meandering channel. The term“binding” refers to the interaction between a corresponding pair ofmolecules that exhibit mutual affinity or binding capacity, typicallyspecific or non-specific binding or interaction, including biochemical,physiological, and/or pharmaceutical interactions. Biological bindingdefines a type of interaction that occurs between pairs of moleculesincluding proteins, nucleic acids, glycoproteins, carbohydrates,hormones and the like. Specific examples include antibody/antigen,antibody/hapten, enzyme/substrate, enzyme/inhibitor, enzyme/cofactor,binding protein/substrate, carrier protein/substrate,lectin/carbohydrate, receptor/hormone, receptor/effector, complementarystrands of nucleic acid, protein/nucleic acid repressor/inducer,ligand/cell surface receptor, virus/ligand, etc.

In some cases, a heterogeneous reaction (or assay) may take place in thechannel; for example, a binding partner may be associated with a surfaceof a channel, and the complementary binding partner may be present inthe fluid phase. The term “binding partner” refers to a molecule thatcan undergo binding with a particular molecule. Biological bindingpartners are examples; for instance, Protein A is a binding partner ofthe biological molecule IgG, and vice versa. Likewise, an antibody is abinding partner to its antigen, and vice versa. In other cases, ahomogeneous reaction may occur in the channel. For instance, bothbinding partners can be present in the fluid phase (e.g., in two-fluidlaminar flow system). Non-limiting examples of typical reactions thatcan be performed in a meandering channel system include chemicalreactions, enzymatic reactions, immuno-based reactions (e.g.,antigen-antibody), and cell-based reactions.

In one embodiment, a signal produced by the reaction is homogenous overthe entire meandering region. In other words, a signal produced in afirst segment of the meandering channel is similar to a signal producedin a second, adjacent segment of the channel, which is similar to thesignal produced in all additional segments of the meandering channel(e.g., all 26 segments of FIG. 1). Such a signal can enable a detectorto be positioned over any portion of the meandering region and allowsimilar signals to be detected by the detector, as shown earlier in FIG.1B and as described in Example 4. In another embodiment, the signal maybe homogeneous over only portions of the meandering region, and one ormore detectors may detect different signals within each of the portions.For instance, a signal produced in a first segment of the meanderingchannel may be similar to a signal produced in a second, adjacentsegment of the channel, but this signal may be different from the signalproduced in, e.g., the 7^(th) and 8^(th), segments of the meanderingchannel. In yet another embodiment, the signal may generate a gradientover the meandering region, e.g., such that the extent of the reactionvaries depending on the location within the channel (e.g., the signal isdifferent in each segment of the channel).

In some embodiments, a meandering channel may be used in combinationwith an amplification system that increases the amount of signal formedin the channel, such as amplification by silver enhancement.Additionally and/or alternatively, precipitating dyes or other materials(e.g., fluorescent molecules or chemiluminescent species) that can forman optically-detectable layer can be used in combination with ameandering channel. For instance, a sample can be flowed over a surfaceassociated with a prospective binding partner of a sample component. Theassay can be performed in a meandering channel of a microfluidic deviceallowing the sample to be flowed over a binding partner, for example, anantigen. Any antigen-antibody complex that forms may be associated witha metal colloid (e.g., a gold colloid) that provides a catalytic surfacefor the deposition of an opaque material, such as a layer of metal(e.g., silver), on a surface of the channel. Therefore, ifantibody-antigen binding occurs in the microfluidic channel, the flowingof a metal precursor through the channel can result in the formation ofan opaque layer (i.e., a substance that interferes with thetransmittance of light at one or more wavelengths), such as a silverlayer, due to the presence of the catalytic metal colloid associatedwith the antibody-antigen complex (FIG. 8). Any opaque layer that isformed in the microfluidic channel can be detected optically, forexample, by measuring a reduction in light transmittance through aportion of the microfluidic channel compared to a portion of the channelthat does not include the antibody or antigen. The opaque layer mayprovide an increase in assay sensitivity when compared to techniquesthat do not form an opaque layer. Examples of this amplification systemare described in Examples 2 and 3, and in further detail inInternational Patent application Serial No.: PCT/US04/043585 by Sia etal., filed Dec. 29, 2004, which is incorporated herein by reference,which is herein incorporated by reference.

FIG. 3 shows the results of heterogeneous immunoassays that wereperformed using a device (similar to microchip 7 in FIG. 2A) thatincluded several meandering regions (see Examples 2 and 3 for moredetails). In FIG. 3A, substrate 200 (the bottom portion of the device)contains five reaction regions, 210, 215, 220, 225, and 230, in theshape of the meandering channels of the device. As expected, regionshaving higher concentrations of reactant gave larger signals (e.g.,region 210) compared to regions containing lower concentrations ofreactant (e.g., region 230). These results are summarized in FIGS. 4 and5 for reactions on substrates 201 and 202, respectively.

In some cases, a microchip having meandering regions may be used incombination with off-chip instrumentation (e.g., a detector). Themeandering regions can be shaped and configured to fit the geometry of aspecific instrument, such as a 96-well plate reader. FIG. 3A showsseveral regions (210, 215, 220, 225, and 230) that are spaced apartaccording to the spacing of the wells in a 96-well plate. Thisconfiguration allows the meandering regions to be used in combinationwith a 96-well reader, which can optically detect several reactions(i.e., in regions 210, 215, 220, 225, and 230) on the microchipsimultaneously.

Fluids may be introduced into meandering channels using any suitablemethod, such as by pipet, syringe, syringe pump, vacuum, or fluidcartridge. In one embodiment, a vessel containing two or more distinctfluids, separated by a third fluid that is immiscible with both, is usedto introduce fluids into a meandering channel. As illustrated in FIG. 9,vessel 600 may be a tube 610 having a longitudinal cross-section thatincludes a reagent solution plug 620, followed by an air plug 630,followed by a rinse solution plug 640. An additional air plug 650 mayseparate the first rinse solution plug from second rinse solution plug660. The ends of the tube 670 and 680 may be sealed, for example, toretain the plugs and to prevent contamination from external sources. Theliquid plugs may retain their relative positions in the tube and may beprevented from contacting each other by the interspaced air plugs. Thetube dimensions and materials of construction may be chosen to helpfluid plugs retain their position and remain unmixed. The vessel isdescribed in more detail in International Patent application Serial No.:PCT/US 05/03514 by Linder et al., filed Jan. 1, 26, 2005, which isincorporated herein by reference.

Reagents and other fluids may be stored for extended lengths of time inthe vessel. For example, reagents may be stored for greater than 1 day,1 week, 1 month or 1 year. By preventing contact between fluids, fluidscontaining components that would typically react or bind with each otherare prevented from doing so, while being maintained in a continuouschamber.

Fluids may be transferred from the vessel to be used in a process, forexample, to participate in a reaction or assay. Fluids may betransferred from the vessel by applying pressure or vacuum afterremoving or piercing the seal at ends 670 and 680. In other embodiments,the vessel need not be sealed and fluid flow can be started by applyingan external force, such as a pressure differential. One end of thevessel, for example, end 670, can be in or can be placed in fluidcommunication with another device that will receive the fluids from thevessel. Such a device may include, for example, a reaction site of areactor or an assay, which can including a meandering channel region.

A vessel containing fluid plugs may be put in fluid communication with areaction site and fluids may be flowed from the vessel to the reactionsite. For instance, the fluids may be flowed to a microfluidicimmunoassay, some embodiments of which are described herein. The vesselcontaining the fluid plugs may be separate from a device including thereaction site or may be part of the same platform. Fluid may be flowedto the reaction site by, for example pushing or pulling the fluidthrough the vessel. Fluids can be pushed to the reaction site using, forexample, a pump, syringe, pressurized vessel, or any other source ofpressure. Alternatively, fluids can be pulled to the reaction site byapplication of vacuum or reduced pressure on a downstream side of thereaction site. Vacuum may be provided by any source capable of providinga lower pressure condition than exists upstream of the reaction site.Such sources may include vacuum pumps, venturis, syringes and evacuatedcontainers.

In one set of embodiments, a vessel may contain fluid plugs in linearorder so that as fluids flow from the vessel to a reaction site they aredelivered in a predetermined sequence. For example, an assay mayreceive, in series, an antibody fluid, a rinse fluid, a labeled-antibodyfluid and a rinse fluid. By maintaining an immiscible fluid (aseparation fluid) between each of these assay fluids, the assay fluidscan be delivered in sequence from a single vessel while avoiding contactbetween any of the assay fluids. Any immiscible fluid that is separatingassay fluids may be applied to the reaction site without altering theconditions of the reaction site. For instance, if antibody-antigenbinding has occurred at a reaction site, air can be applied to the sitewith minimal or no effect on any binding that has occurred.

In one embodiment, at least two fluids may be flowed in series from acommon vessel, and a component of each fluid may participate in a commonreaction. As used herein, “common reaction” means that at least onecomponent from each fluid reacts with the other after the fluids havebeen delivered from the vessel, or at least one component from eachfluid reacts with a common component and/or at a common reaction siteafter being delivered from the vessel. For example, a component of thefirst fluid may react with a chemical or biological entity that isdownstream of the vessel. A chemical or biological entity may form areaction site and may be, for example, a sample, a biological orchemical compound, a cell, a portion of a cell, a surface or asubstrate. The chemical or biological entity may be fixed in position ormay be mobile. A component from the second fluid may then react and/orassociate with the component from the first fluid that has reacted withthe downstream chemical or biological entity, or it may react orassociate with the chemical or biological entity itself. Additionalfluids may then be applied, in series, to the biological or chemicalentity to effect additional reactions or binding events or as indicatorsor signal enhancers.

Pre-filling of the vessel with reagents may allow the reagents to bedispensed in a predetermined order for a downstream process. In caseswhere a predetermined time of exposure to a reagent is desired, theamount of each fluid in the vessel may be proportional to the amount oftime the reagent is exposed to a downstream reaction site. For example,if the desired exposure time for a first reagent is twice the desiredexposure time for a second reagent, the volume of the first reagent inthe vessel may be twice the volume of the second reagent in the vessel.If a constant pressure differential is applied in flowing the reagentsfrom the vessel to the reaction site, and if the viscosity of the fluidsis the same or similar, the exposure time of each fluid at a specificpoint, such as a reaction site, may be proportional to the relativevolume of the fluid. Factors such as vessel geometry, pressure orviscosity can also be altered to change flow rates of specific fluidsfrom the vessel.

When designing a microfluidic device for use with a particularcomponent, e.g., a fluid vessel as described above, certain aspects ofthe design may have to be taken into consideration. For instance, thedimensions of the meandering channel (e.g., channel size and aspectratio of the channel) may be chosen to avoid the retention of airbubbles within the device, since air bubbles can cause inaccuratedetection of a signal. This can configuration may allow multiplemeandering regions to be positioned in series. Generally, channels withlarger cross-sectional dimensions avoid the trapping of air bubbles morethan channels with smaller cross-sectional dimensions, since largedimensions allow the bubbles to have less surface area in contact withthe walls of the channel. Channels having a width of greater than 25 μm,greater than 50 μm, greater than 75 μm, greater than 100 μm, or greaterthan 200 μm may be suitable for avoiding the trapping of air bubbles,depending aspects such as the size/volume of the air pockets introducedinto the device, the fluids surrounding the air bubbles, and thehydrophilicity/hydrophobicity of the walls of the channel.

In another embodiment, a wide channel or a chamber having a surface areaon the order of millimeters squared (mm²) is used a reaction site on amicrofluidic chip. For instance, the width of the channel or chamber maybe greater than or equal to 0.5 mm, greater than or equal to 1 mm,greater than or equal to 2 mm, greater than or equal to 5 mm, greaterthan or equal to 10 mm. The area covered by the wide channel is designedto be relatively large (e.g., on the order of mm²) compared toconventional microfluidic systems, so that a wide area can be used fordetection and so the total amount of signal that can be detected isincreased. For instance, the surface area covered by the wide channelmay be greater than or equal to 0.5 mm², greater than or equal to 1 mm²,greater than or equal to 2 mm², greater than or equal to 5 mm², greaterthan or equal to 10 mm², or greater than or equal to 50 mm². As shown inFIG. 7, fluidic chip 400 comprises several wide channels 410. The areathat wide channel 410 covers is defined by the square area given bydimension 420 times (x) dimension 430.

Similar to a meandering channel, a wide channel or chamber havingdimensions on the order of square-millimeters allows a user to read asignal (e.g., a change in color, turbidity, opacity, agglutination,etc.) directly from the microchip, and/or, to align a detector over awide portion of the channel, rather than over a narrow (e.g., μm)portion of the channel, as is typical in conventional microfluidicsystems. The large area of wide channel 410 also allows it to becompatible with simplified optical detectors (as discussed below), aswell as with conventional spectrophotometers and optical readers (e.g.,96-well plate readers).

Wide channel 410 has an upper surface, a lower surface, and mayoptionally comprise posts (e.g., posts 440, 450, and/or 460) disposedbetween the upper and lower surfaces. As shown in FIG. 7, posts 440,450, and/or 460 may have different shapes and/or configurations. In somecases, the posts are used to give support to the upper and lowersurfaces. For instance, wide channels fabricated in an elastomer mayrequire posts to prevent the upper surface and lower surface fromcontacting each other. Posts can also be used in microfluidic systemsfor other purposes such as for mixing and filtering.

Wide channel 410 has at least one cross-sectional dimension in themicron range so that it can retain its advantageous qualities ofmicro-scale geometry (e.g., laminar flow, fast reaction times, smallvolumes, etc.). For example, the cross-sectional dimension may be lessthan 1 mm, less than 500 μm, less than 250 μm, less than 100 μm, lessthan 50 μm, or less than 25 μm. A cross-sectional dimension in themicron range also allows transport of fresh solution over the surface ofthe channel without the use of large amounts of fluid.

In some cases, it is advantageous to use a wide channel region over ameandering channel region, e.g., for performing a reaction or assay. Forinstance, when mixing is desired, e.g., mixing two or more solutions ortwo or more components within a solution, it may be suitable to use awide channel region (with or without posts). In other cases, it isadvantageous to use a meandering channel region over a wide channelregion. For example, if mixing is not desired, or if gas bubbles areintroduced into the fluidic system, meandering channels may be moresuitable. In some instances, a microfluidic device comprises both a widechannel region and a meandering region, e.g., when mixing is desired inone portion of the device but not desired in another.

A variety of determination techniques may be used. Determinationtechniques may include optically-based techniques such as lighttransmission, light absorbance, light scattering, light reflection andvisual techniques. Determination techniques may also includeluminescence techniques such as photoluminescence (e.g., fluorescence),chemiluminescence, bioluminescence, and/or electrochemiluminescence.Those of ordinary skill in the art know how to modify microfluidicdevices in accordance with the determination technique used. Forinstance, for devices including chemiluminescent species used fordetermination, an opaque and/or dark background may be preferred. Fordetermination using metal colloids, a transparent background may bepreferred.

In some embodiments, determination techniques may measure conductivity.For example, microelectrodes placed at opposite ends of a portion of amicrofluidic channel may be used to measure the deposition of aconductive material, for example an electrolessly deposited metal. As agreater number of individual particles of metal grow and contact eachother, conductivity may increase and provide an indication of the amountof conductor material, e.g., metal, that has been deposited on theportion. Therefore, conductivity or resistance may be used as aquantitative measure of analyte concentration.

Another analytical technique may include measuring a changingconcentration of a precursor from the time the precursor enters themicrofluidic channel until the time the precursor exits the channel. Forexample, if a silver nitrate solution is used, a silver sensitiveelectrode may be capable of measuring a loss in silver concentration dueto the deposition of silver in a channel as the precursor passes throughthe channel.

Different optical detection techniques provide a number of options fordetermining assay results. In some embodiments, the measurement oftransmission or absorbance means that light can be detected at the samewavelength at which it is emitted from a light source. Although thelight source can be a narrow band source emitting at a single wavelengthit may also may be a broad spectrum source, emitting over a range ofwavelengths, as many opaque materials can effectively block a wide rangeof wavelengths. The system may be operated with a minimum of opticaldevices (e.g., a simplified optical detector). For instance, thedetermining device may be free of a photomultiplier, may be free of awavelength selector such as a grating, prism or filter, may be free of adevice to direct or columnate light such as a columnator, or may be freeof magnifying optics (e.g., lenses). Elimination or reduction of thesefeatures can result in a less expensive, more robust device.

In one embodiment, the light source can be pulse modulated, for example,at a frequency of 1,000 Hz. To match the pulse modulated light source, adetector may include a filter operating at the same frequency. By usinga pulse modulated light source it has been found that the system can beless sensitive to extrinsic sources of light. Therefore, the assay mayrun under various light conditions, including broad daylight, that mightmake it impractical to use existing techniques. Experimental resultsindicate that by using a pulse modulated light source and filter,results are consistent regardless of the light conditions under whichthe test is run.

The light source may be a laser diode. For example, an InGaAlP redsemiconductor laser diode emitting at 654 nm may be used. Thephotodetector may be any device capable of detecting the transmission oflight that is emitted by the light source. One type of photodetector isan optical integrated circuit (IC) including a photodiode having a peaksensitivity at 700 nm, an amplifier and a voltage regulator. If thelight source is pulse modulated, the photodetector may include a filterto remove the effect of light that is not at the selected frequency.

Though in some embodiments, systems of the invention may bemicrofluidic, in certain embodiments, the invention in not limited tomicrofluidic systems and may relate to other types of fluidic systems.“Microfluidic,” as used herein, refers to a device, apparatus or systemincluding at least one fluid channel having a cross-sectional dimensionof less than 1 mm, and a ratio of length to largest cross-sectionaldimension of at least 3:1. A “microfluidic channel,” as used herein, isa channel meeting these criteria.

The “cross-sectional dimension” of the channel is measured perpendicularto the direction of fluid flow. Most fluid channels in components of theinvention have maximum cross-sectional dimensions less than 2 mm, and insome cases, less than 1 mm. In one set of embodiments, all fluidchannels containing embodiments of the invention are microfluidic orhave a largest cross sectional dimension of no more than 2 mm or 1 mm.In another embodiment, the fluid channels may be formed in part by asingle component (e.g., an etched substrate or molded unit). Of course,larger channels, tubes, chambers, reservoirs, etc. can be used to storefluids in bulk and to deliver fluids to components of the invention. Inone set of embodiments, the maximum cross-sectional dimension of thechannel(s) containing embodiments of the invention are less than 500microns, less than 200 microns, less than 100 microns, less than 50microns, or less than 25 microns. In some cases the dimensions of thechannel may be chosen such that fluid is able to freely flow through thearticle or substrate. The dimensions of the channel may also be chosen,for example, to allow a certain volumetric or linear flowrate of fluidin the channel. Of course, the number of channels and the shape of thechannels can be varied by any method known to those of ordinary skill inthe art. In some cases, more than one channel or capillary may be used.For example, two or more channels may be used, where they are positionedinside each other, positioned adjacent to each other, positioned tointersect with each other, etc.

A “channel,” as used herein, means a feature on or in an article(substrate) that at least partially directs the flow of a fluid. Thechannel can have any cross-sectional shape (circular, oval, triangular,irregular, square or rectangular, or the like) and can be covered oruncovered. In embodiments where it is completely covered, at least oneportion of the channel can have a cross-section that is completelyenclosed, or the entire channel may be completely enclosed along itsentire length with the exception of its inlet(s) and outlet(s). Achannel may also have an aspect ratio (length to average cross sectionaldimension) of at least 2:1, more typically at least 3:1, 5:1, or 10:1 ormore. An open channel generally will include characteristics thatfacilitate control over fluid transport, e.g., structuralcharacteristics (an elongated indentation) and/or physical or chemicalcharacteristics (hydrophobicity vs. hydrophilicity) or othercharacteristics that can exert a force (e.g., a containing force) on afluid. The fluid within the channel may partially or completely fill thechannel. In some cases where an open channel is used, the fluid may beheld within the channel, for example, using surface tension (i.e., aconcave or convex meniscus).

A microfluidic device of the invention can be fabricated of any materialsuitable for forming a microchannel. Non-limiting examples of materialsinclude polymers (e.g., polystyrene, polycarbonate,poly(dimethylsiloxane), and a cyclo-olefin copolymer (COC)), glass, andsilicon. Those of ordinary skill in the art can readily select asuitable material based upon e.g., its rigidity, its inertness to (i.e.,freedom from degradation by) a fluid to be passed through it, itsrobustness at a temperature at which a particular device is to be used,and/or its transparency/opacity to light (i.e., in the ultraviolet andvisible regions).

In some instances, the body is comprised of a combination of two or morematerials, such as the ones listed above. For instance, the channels ofthe device may be formed in a first material (e.g.,poly(dimethylsiloxane)), and a substrate that is formed in a secondmaterial (e.g., polystyrene) may be used as the base to seal thechannels.

In some cases, the device is fabricated using rapid prototyping and softlithography. For example, a high resolution laser printer may be used togenerate a mask from a CAD file that represents the channels that makeup the fluidic network. The mask may be a transparency that may becontacted with a photoresist, for example, SU-8 photoresist (MicroChem),to produce a negative master of the photoresist on a silicon wafer. Apositive replica of PDMS may be made by molding the PDMS against themaster, a technique known to those skilled in the art. To complete thefluidic network, a flat substrate, for example, a glass slide, siliconwafer, or polystyrene surface may be placed against the PDMS surface andmay be held in place by van der Waals forces, or may be fixed to thePDMS using an adhesive. To allow for the introduction and receiving offluids to and from the network, holes (for example 1 millimeter indiameter) may be formed in the PDMS by using an appropriately sizedneedle. To allow the fluidic network to communicate with a fluid source,tubing, for example of polyethylene, may be sealed in communication withthe holes to form a fluidic connection. To prevent leakage, theconnection may be sealed with a sealant or adhesive such as epoxy glue.

The following examples are intended to illustrate certain embodiments ofthe present invention, but are not to be construed as limiting and donot exemplify the full scope of the invention.

EXAMPLE 1 Fabrication and Design of a Meandering Channel System

A method for fabricating and designing a meandering channel system isdescribed. Using rapid prototyping techniques, a master of SU8-50photoresist was produced on a silicon wafer. The master was used toreplicate the negative pattern in PDMS. The height of the photoresistfeatures was 52 μm. The master was silanized with(tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane (ABC-R, Germany).PDMS (Silgard 184, Dow Corning, from Distrelec, Nanikon, Switzerland)was mixed according to the manufacturer's instructions and poured ontothe master. After polymerization (4 hours, 65°), the PDMS replica waspeeled off the master and access holes were punched out of the PDMSusing brass tubing with sharpened edges (1.5 mm in diameter).

The layout of the channel system was designed with a computer-aideddesign (CAD) program and is illustrated in FIG. 2A. The channels were120 μm in width, and the area of the meandering region covered thesurface of a well in a 96-well plate. The positions of the meanderingregions relative to one other were the same as that of wells in a96-well plate. This arrangement enabled the PDMS replica to be placedonto a “NUNC plate”, a plate having the same dimensions as the 96-wellplate but without the wells. This arrangement was chosen so that thequantification of the signal generated inside the meandering regioncould be performed with a standard ELISA plate reader, as well as withother types of optical detectors.

EXAMPLE 2 Immunoassay Experiment Using Silver Enhancement

An immunoassay experiment was performed using the meandering channelsystem as described in Example 1. In preparation for the assay, thesurface of a NUNC plate was spotted with an array of droplets of humanIgG (50 μg/mL in PBS). Each droplet covered a circle of ˜1 cm indiameter, each droplet being aligned with the location of a microfluidicmeandering region (based on a 96-well plate as the template). Theprotein solution was left to incubate overnight at 4° C. The plates wererinsed with deionized water, dried with a stream of nitrogen, and thePDMS replica was sealed (conformally, i.e., without plasma oxidation) tothe NUNC plate. A 96-well plate was used as a template to align themeandering regions to the spots of human IgG on the NUNC plate. Themicrochannels were filled with a solution of 0.05% Tween inphosphate-buffered saline (PBS), which allowed blocking of the surfaceof the microchannels for 1 hour at 4° C.

An immunoassay was performed using fluid vessels (in the form of atube), as described previously. The buffer used for the assay was 0.05%Tween in PBS. Sections of PE60 tubing were prepared, and filled with asuccession of reagents, each reagent being separated by pockets of air(e.g., air bubbles). The section of tubing was filled with 5 cm (23 μL)of labeled anti-human IgG (labeled with either gold colloids, or withhorseradish peroxidase (Example 3)), prepared at various concentrationsby serial dilution with buffer, followed by three 3-mm long plugs ofbuffer, and a 10-mm long plug of buffer. The tubing was fitted into aninlet of the microchip, the inlet in fluid communication with themeandering channels. A negative pressure (−15 kPa) applied at the outletof the channel drew all of the reagents from the cartridge and thoughthe meandering regions of the microchip. When all of the reagents hadpassed through the meandering regions, the empty cartridge wasdisconnected from the microchip and an amplification solution waspipetted into the inlet of the channel while the negative pressure wasmaintained.

For amplification targeting anti-human IgG labeled with gold colloids(Sigma, St. Louis), a solution for electroless silver deposition (Sigma)was used as the amplification solution. The solution was freshlyprepared from the two components (silver nitrate and hydroquinone),mixed in equal amounts. The silver solution remaining at the inlet wasexchanged with another freshly prepared solution every 6 minutes.Amplification was continued until a clear signal was observable by thenaked eye (18 minutes), as shown in FIGS. 3A and 3B. Channels containinghigher concentrations of reagent (gold-labeled anti-human IgG) gavelarger signals. In FIG. 3A, the dilution of gold-labeled anti-human IgGwas 1:50 (region 210), 1:500 (region 215), 1:1500 (region 220), 1:5000(region 225), and 1:50,000 (region 23). In FIG. 3B, the dilution ofgold-labeled anti-human IgG was 1:50 (region 250), 1:500 (region 255),1:5000 (region 260), and 1:50,000 (region 265).

The channels were rinsed with PBS, and the PDMS structure containing themeandering channels was removed the from the NUNC plate. The surface ofthe PDMS was dried using a stream of nitrogen. Optical densitymeasurements (of several reactions, obtained simultaneously) with anELISA reader showed results that were consistent with the intensitiesvisible by eye. A sigmoidal calibration curve was obtained for allsubstrates. FIG. 4 shows a calibration curve obtained for the anti-humanIgG assay with amplification by silver enhancement, based on theexperiment shown in FIG. 3B.

EXAMPLE 3 Immunoassay Experiment Using Enzymatic Amplification andPrecipitating Dyes

An immunoassay experiment was performed using the meandering channelsystem of Example 1, and following similar procedures as described inExample 2, but with enzymatic amplification and the precipitating dyeDAB instead of silver enhancement.

For amplification with horseradish peroxidase labeled anti-human IgG(Sigma), a freshly-prepared solution of DAB (diaminobenzidine) (Fluka,Switzerland) was used. DAB was dissolved in DMSO at a concentration of10 mg/mL to obtain a stock solution of DAB. The working solution wasprepared by diluting 100-fold the stock solution of DAB in PBS, and byadding hydrogen peroxide (30%, Rockwood, France) to obtain a finaldilution of 1:3000 in hydrogen peroxide. The amplification solution wasdrawn into the meandering region until a signal was visible by eye(i.e., 30 minutes), as shown in FIG. 3C. In FIG. 3C, the dilution oflabeled anti-human IgG was 1:10 (region 270), 1:50 (region 275), 1:100(region 280), 1:500 (region 285), 1:2,500 (region 290), and 1:12,500(region 295).

FIG. 5 shows the result of the assay shown in FIG. 3C, using an ELISAplate reader as the detector. Although the amplification was carried outfor 30 minutes (compared to 18 minutes for the silver enhancementamplification), the intensities were much smaller using DABamplification than those obtained with the silver chemistry. Underconditions for DAB amplification, the linear range of the calibrationcurve was close to 1000-fold dilution of anti-human IgG. Both silver andDAB amplification chemistries could thus be used for the quantificationof heterogeneous assays.

EXAMPLE 4 Alignment of the Meandering Channels

The data in FIG. 6 were obtained with only mm-range precision ofalignment between the meandering regions and the light beam of thedetector (in this example, an ELISA reader). Curves 300 and 310 showthat after rotating the substrate 180 degrees (around the z-axis)relative to the ELISA reader, similar signals were detected. Both setsof results were within the expected results for the assay. These resultsindicate that the width of the light beam of the ELISA reader wassufficiently larger than the width of the microchannel, and encompassedseveral segments of the meandering channel. In this configuration, onlythe alignment of the light beam to the mm-sized meandering region wasnecessary, instead of careful alignment to an individual section of themeandering region. The signal detected from each of the meanderingregions remained unchanged, despite the loss of initial alignmentbetween the microchannels and the beam of light.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of”, when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

What is claimed is:
 1. A method comprising: carrying out a chemicaland/or biological reaction in a meandering region of a microfluidicchannel; exposing the meandering region to light; and measuring a signalaveraged over more than one adjacent segments of the microfluidicchannel in the meandering region.
 2. A method comprising: passing afluid over a surface of a meandering region of a microfluidic channel,wherein at least a portion of the microfluidic channel in the meanderingregion has a width of less than 0.5 mm; accumulating an opaque materialin the meandering region of the microfluidic channel; and measuring asignal averaged over more than one adjacent segments of the microfluidicchannel in the meandering region.
 3. A system comprising: a microfluidicchannel comprising a meandering region defined by an area of at least0.5 mm², wherein at least a portion of the area of the meandering regioncomprises an optical detection pathway, and wherein at least a portionof the microfluidic channel in the meandering region has a cross-sectionthat is completely enclosed; and a detector adapted and arranged tomeasure a signal averaged over more than one adjacent segments of themicrofluidic channel in the meandering region.
 4. A method as in claim1, wherein the signal is a light transmittance signal or a lightabsorbance signal.
 5. A method as in claim 1, wherein determining thesignal comprises using an optical system that does not comprise anoptical magnifying component.
 6. A method as in claim 1, wherein a firstportion of the signal, measured from a first segment of the microfluidicchannel in the meandering region, is substantially the same as a secondportion of the signal, measured from a second, adjacent segment of themicrofluidic channel in the meandering region.
 7. A method as in claim1, wherein a portion of the signal, measured from a first segment of themicrofluidic channel in the meandering region, is substantially the sameas a portion of the signal measured from at least 5 adjacent segments ofthe microfluidic channel in the meandering region.
 8. A method as inclaim 1, wherein at least a portion of the microfluidic channel in themeandering region has a width of less than 500 microns.
 9. A method asin claim 1, wherein at least a portion of the microfluidic channel inthe meandering region has a width of less than 250 microns.
 10. A methodas in claim 1, wherein the chemical and/or biologic reaction is abinding event between at least two binding partners, and wherein atleast one of the binding partners comprises an antibody.
 11. A method asin claim 1, comprising allowing a sample component to bind with abinding partner disposed on a surface of the microfluidic channel in themeandering region.
 12. A method as in claim 11, wherein a metal colloidis associated with the sample component.
 13. A method as in claim 12,comprising flowing a metal solution over the surface to form a metalliclayer.
 14. A method as in claim 1, comprising forming an opaque materialin the meandering region of the microfluidic channel, and quantitativelydetermining the opacity of the opaque material.
 15. A method as in claim14, wherein quantitatively determining the opacity of the opaquematerial comprises measuring light transmission or light absorbancethrough the opaque material.
 16. A method as in claim 1, wherein themeandering region is a first meandering region and the signal averagedover more than one adjacent segments of the microfluidic channel in thefirst meandering region is a first signal, wherein the first meanderingregion is in fluid communication with and positioned in series withrespect to a second meandering region comprising a second microfluidicchannel, and wherein the method comprises measuring a second signalaveraged over more than one adjacent segments of the second microfluidicchannel.
 17. A method as in claim 1, wherein a first segment of themicrofluidic channel in the meandering region is spaced apart from asecond, adjacent segment of the microfluidic channel in the meanderingregion by a distance of less than the average width of the microfluidicchannel in the meandering region.
 18. A method as in claim 1, wherein afirst segment of the microfluidic channel in the meandering region isspaced apart from a second, adjacent segment of the microfluidic channelin the meandering region by a distance of less than 2 times the averagewidth of the microfluidic channel in the meandering region.
 19. A methodas in claim 1, comprising flowing in series in the microfluidic channela predetermined sequence of fluid plugs including first, second andthird fluids, wherein the first and second fluids are separated by thethird fluid which is immiscible with both the first and second fluids.20. A method as in claim 19, wherein the first and second fluids areliquids and the third fluid is a gas.
 21. A method as in claim 20,wherein the first and/or second fluids is a rinse solution.
 22. A methodas in claim 21, wherein the first fluid is a rinse solution and thesecond fluid is a metal solution.
 23. A method as in claim 1, whereinthe signal is substantially homogeneous over more than one adjacentsegments of the microfluidic channel.
 24. A method as in claim 1,wherein the signal is substantially homogeneous over all of the segmentsof the microfluidic channel in the first meandering region.
 25. A methodas in claim 23, wherein the meandering region has an area of at least0.5 mm², and wherein the exposing step involves exposing at least 50% ofthe area of the meandering region to light.